This paper evaluates a novel high variability phonetic training paradigm that involves presenting spoken words in adverse conditions. The effectiveness, generalizability, and longevity of this high variability phonetic training in adverse conditions was evaluated using English phoneme contrasts in three experiments with Malaysian multilinguals. Adverse conditions were created by presenting spoken words against background multi-talker babble. In Experiment 1, the adverse condition level was set at a fixed level throughout the training and in Experiment 2 the adverse condition level was determined for each participant before training using an adaptive staircase procedure. To explore the effectiveness and sustainability of the training, phonemic discrimination ability was assessed before and immediately after training (Experiments 1 and 2) and 6 months after training (Experiment 3). Generalization of training was evaluated within and across phonemic contrasts using trained and untrained stimuli. Results revealed significant perceptual improvements after just three 20-minute training sessions and these improvements were maintained after 6 months. The training benefits also generalized from trained to untrained stimuli. Crucially, perceptual improvements were significantly larger when the adverse conditions were adapted before each training session than when it was set at a fixed level. As the training improvements observed here are markedly larger than those reported in the literature, this indicates that the individualized phonetic training regime in adaptive adverse conditions (HVPT-AAC) is highly effective at improving speech perception. Non-native English speakers have often difficulties discriminating phonemic contrasts that involve phoneme categories that are not present in their native language. For example, Japanese speakers have difficulties distinguishing between the English phonemes /r/ and /l/ –4, MandaA large number of studies have investigated perceptual phonetic training methods to improve speech perception in the non-native language using either synthetic e.g., –12) or n or n12])One of the most successful perceptual training methods was introduced by Lively and colleagues who showed the beneficial effects of training with highly variable material (later referred to as High Variability Phonetic Training or HVPT) with Japanese native speakers learning the English /r/-/l/ contrast –15. A tyA large number of studies have demonstrated that compared to native speakers, speech perception in adverse conditions is particularly challenging for non-native speakers e.g., , 27). Ad. Ad27]).A series of studies conducted by Bradlow and colleagues showed that the non-native deficit in speech-in-noise perception may be attributed to their perceptual strategy that is different from the native speakers. An example of a native speakers’ strategic perceptual approach can be seen in the clear speech effect, which is the intelligibility difference between clear speech and normal conversational speech. In clear speech production, the acoustic salience of the speech signals is enhanced. Native speakers in the study by Bradlow and Bent  benefiteWhen speech signals are degraded by an informational masker , non-native speakers’ difficulty in understanding speech can be attributed to the greater interference of the native phonetic system when there is ambiguity during second language (L2) processing , the increasing cognitive load in non-native speakers when processing two sources of non-native auditory information, or the non-native speakers’ inexperience in separating the signal from the masker language , 36, espAlthough daily life conversations are often held in suboptimal or adverse conditions, very few phonetic training studies have been conducted with non-native normal-hearing listeners in difficult listening conditions. As far as we are aware, only Jamieson and Morosan  used bacTraining in noise, however, is common in perceptual training studies involving hearing-impaired listeners. In some of these studies, speech-shaped noise was used to simulate hearing loss in normal-hearing native listeners . A studInterestingly, novel research conducted by Hazan and colleagues  focusedThe purpose of the present experiments was to examine the effectiveness of high variability phonetic training in adaptive adverse conditions (HVPT-AAC). The initial concept of HVPT-AAC was developed by the late Richard Pemberton, Kathy Conklin, Nicola Pitchford, and Walter van Heuven at the University of Nottingham. The implementation of HVPT-AAC for the research presented here was developed by the last two authors of this paper. HVPT-AAC uses natural speech stimuli  spoken by multiple speakers . Critical features of HVPT-AAC include presentation of spoken words in background noise in the form of multi-talker babble (adverse conditions) and an adaptive level of adverse conditions whereby an optimal training level can be set for each listener through an adaptive staircase procedure (see Experiment 2 for further details). Adverse conditions in perceptual training increases the speech perception difficulties not only for non-native speakers but also for highly proficient non-native speakers and native speakers whose disadvantage in speech perception may only manifests itself in the presence of noise  and the effectiveness of adapting the level of adverse conditions in HVPT (HVPT-AAC) versus a fixed level of adverse conditions (Experiment 2). The longevity of training in adverse conditions were investigated in Experiment 3.In this experiment we investigated whether perceptual training in two different levels of adverse conditions modulated the training results of non-native speakers, and whether the training generalized to untrained stimuli and untrained contrasts. Two levels of adverse conditions were created by manipulating the volume of the multi-talker babble (low vs. high) relative to the target stimulus volume level. We expected to see greater training benefits in the more adverse training condition (high volume) because listeners have to learn how to engage more effectively the cognitive processes used in the task  when target auditory information is masked in the higher level of adverse conditions.A group of 28 participants  who spoke Mandarin as their first language (L1) were recruited from the University of Nottingham, Malaysia Campus. All participants reported to have normal or corrected to normal vision, and they had no history of any hearing, speech, or reading problems. Participants were paid for their participation.Participants completed a questionnaire to obtain information about their language background. The students' English Language test scores as required for admission onto their university course were converted to IELTS standard scores using the University of Nottingham English language qualification equivalencies. All participants spoke Malaysian English and Malay as their L2, and learnt both languages from a young age . All participants had learnt the two languages for at least 11 years through formal education and were pursuing their tertiary education in English during the study. 22 participants also spoke at least one other Chinese language . In addition, 3 participants reported to have learned a foreign language (Japanese or German) at a low proficiency .Participants were assigned randomly to either the low or high adverse condition groups. Between-subjects t-tests were then conducted to compare their linguistic experience and ability, so that the groups were matched in terms of their self-rated language proficiency, AoA and IELTS score.The mean self-rated English and Mandarin proficiency did not differ between the two groups. Participants also did not differ in terms of their age of English acquisition (AoA) and IELTS standardized test scores.tame–dame) and eight at the final position , and sixteen /ε/-/æ/ minimal pairs . These two phonemic contrasts were chosen because they are difficult for the L1 Mandarin speakers  = 3.84, p < .05). For the training sessions, the sixteen /ε/-/æ/ minimal pairs were split into two sets of stimuli (trained and untrained). Half of the participants were trained with one set of stimuli, whereas the other participants were trained with the other set. Four speakers  with different English accents  recorded the spoken word stimuli. The stimuli were recorded in an Anechoic chamber using an AKG Perception 400 microphone connected to a Presonus FireBox, which was linked to an Apple Macbook Pro. Speech was recorded at 44.1.kHz (16 bit) using Amadeus Pro (version 2). Recordings were edited using Amadeus Pro and the volume of the recordings were normalised by amplifying the sound recordings of each speaker to an average root mean square (RMS) power of -25 dB (200 ms window). Stimuli spoken by the male Southern Irish English speaker were presented in the first training session, stimuli spoken by the female American English speaker were used in the second training session, and the stimuli spoken by the male Irish English speaker were presented in the third training session. The stimuli spoken by the female British English speaker were used in the pre- and post-tests.The stimuli consisted of three groups of English minimal pairs: sixteen /t/-/d/ minimal pairs of which eight differed at the initial position . The siposition ), numberThe background noise consisted of 6-talker babble and was created by combining the audio recording of 6 native English speakers  taken from six BBC Radio 4 interviews in which the interviewees talked about their life and work. The interviewer's voice was edited out and the volume of each speaker was normalised using Amadeus Pro by amplifying the sound to an average root mean square (RMS) power of -25 dB (200 ms window). The resulting 6 audio files were combined into a single 6-talker babble mono audio file  of 6 minutes.During pre-test, post-test and training with a high level of background babble, the multi-talker babble was played continuously during the task at half the stimulus level with a mean signal-to-noise ratio (SNR) of -2.6 dB (range -8.4 to 1.9 dB). During training with a low level of background babble, the multi-talker babble was played at one-tenth of the stimulus level with a mean SNR of 11.3 dB (range 5.6 to 15.9 dB). Praat  was usedAll participants completed five sessions, one session per day. On the first day, participants completed the pre-test, followed by three training sessions spread across the following three consecutive days and then on the final day the participants completed the post-test. A 14-inch laptop (HP EliteBook 8460p) was used to run the training program and a mouse was used to record responses.The pre- and post-tests consisted of two alternative forced-choice (2AFC) identification task. The stimuli  in this task were repeated four times resulting in a total of a hundred and twenty-eight experimental trials. In each trial, the two words of a minimal pair were visually presented side by side at center of the computer screen and at the same time one of the words was presented auditorily. Participants were asked to indicate which word on the computer screen matched with the word they heard. Auditory stimuli were presented at a comfortable listening level set by each participant using Sony Headphones (MDR-NC8/WHI).Participants completed eight practice trials in order to familiarize themselves with the task. Presentation of the minimal pairs was randomized. No feedback was provided after each trial. Only at the end of the practice trials and after each block of 32 experimental trials, the total percentage correct was presented. The pre- and post-tests each took approximately 15 minutes.In the training sessions participants performed a Same-Different word discrimination task with background babble presented either at one-tenth or half of the stimulus level. Participants heard pairs of words and had to decide whether the words were the same or different by clicking on one of the two response buttons ("Same" or "Different") presented on the computer screen using a computer mouse. The second word was played 1000 ms after the first word. Participants received feedback after each response. After a correct response, the response button with the correct answer turned green and then the next trial started. After an incorrect response, the button with the incorrect answer turned red and the correct answer turned green. The word pair was played again before the next trial was presented and no response was needed.Each training session lasted approximately 20 minutes. Participants were instructed to focus on the auditory words they heard and try to ignore the multi-talker babble played in the background. All participants gave written informed consent prior to the start of the experiment.gav and Hedges’ gs) were calculated for significant findings using the spreadsheet provided by Lakens , η2G = 0.02. There was no interaction between the level of background babble and the time of test, indicating that both levels of background babble yielded similar levels of improvement in identification accuracy. Furthermore, there was no interaction between type of contrast and the time of test, which indicates that the training generalized to the stimuli of the untrained contrast /t/-/d/ final.The impact of training in adverse conditions with a low and high fixed level of background babble was examined in a 2 x 2 x 2 mixed ANOVA with the level of background multi-talker babble (low vs. high SNR) as the between-subject factor, and time of test (pre-test vs. post-test) and contrasts  as the within-subject factors. Overall, participants' perceptual performance was 3.0% better in the post-test  than in the pre-test , F = 8.91, p < .01, η2p = 0.26, 95% CI, η2G = 0.04 (5.1% difference). Importantly, there was no interaction between stimulus set (trained vs. untrained /ε/-/æ/) and the time of test, which indicates that the perceptual improvements generalized to the untrained stimuli.The above analysis included trained and untrained stimuli from the /ε/-/æ/ contrast. To assess whether the effect of the trained /ε/-/æ/ stimuli also generalized to untrained /ε/-/æ/ stimuli another mixed ANOVA was conducted , the HVPT training in adverse conditions was able to further improve their overall perceptual performance by 3.0%.Similar to findings from other training studies , 2, 24, The current findings also indicate that learning from the training transferred to a different speaker because the speaker of the post-test spoke a different variety of English . Participants’ performance improved after training regardless of the level of adverse conditions used during training. This suggests that training in adverse conditions improves perceptual performance of non-native speakers and increasing the level of adverse conditions does not seem to influence the training outcomes. However, it is important to note that there were large individual differences in the current study. The percentage identification accuracy in the pre-test varied between 56% to 93%. In this experiment, the level of background multi-talker babble was not adapted to the participants’ individual performance level. Therefore, the participants' auditory system might not have been stressed sufficiently to maximize training benefits.In our second experiment, we examined the impact of HVPT training in adaptive adverse conditions (HVPT-AAC). The level of background multi-talker babble  was determined before each training session using an adaptive staircase procedure. This individually determined SNR was then used in the subsequent training session with stimuli of the same speaker as used in the adaptive staircase procedure.An adaptive staircase procedure is a psychometric method used to measure a person’s sensory capabilities and is used to determine the person’s threshold or limit to detect and discriminate similar and confusable physical stimuli . This prBy combining the strengths of existing training methods and individualized levels of adverse conditions, HVPT-AAC is expected to be more effective than HVPT with a fixed level of background multi-talker babble (as used in Experiment 1) in terms of its training improvements and generalizability. Participants in Experiment 2 had more diverse Malaysian English proficiency levels  to investigate whether the perceptual performance improvements were modulated by Malaysian English proficiency. It was expected that the training would benefit all Malaysian speakers, irrespective to their English proficiency level due to the adaptiveness of the adverse conditions in HVPT-AAC.n = 14), Malay (n = 14) or Malaysian English (n = 11) as their L1. All participants also spoke both Malaysian English and Malay from young age (from 0–7 years old). One L1 Malay speaker was also able to speak Mandarin, but at a very low proficiency level. All participants had normal or corrected to normal vision, and had no history of any hearing, speech and reading disorders. The language background of the participants was assessed with the same language questionnaire as used in Experiment 1  were recruited from the University of Nottingham, Malaysia campus. One participant was excluded due to the loss of a data file. Participants either spoke Mandarin  = 9.47, p < .001 and IELTS scores, F = 3.57, p < .05. However, post-hoc t-tests with Bonferroni correction indicated that L1 Malay and L1 Mandarin participants did not differ in terms of their self-rated English proficiency scores and IELTS sores. For IELTS scores, only L1 Malay participants were found to score significantly lower compared to L1 Malaysian English participants (p < .05). As expected, L1 Malaysian English participants reported a higher level of English proficiency compared to the Malay and Mandarin groups (ps < .01).To examine whether the language groups differed in terms of their English language proficiency, a between-subjects one-way ANOVA was conducted with IELTS scores and the mean self-rated English proficiency. Results revealed that the groups differed significantly in terms of self-rated English proficiency, The design and stimuli were identical to Experiment 1. The two phonemic contrasts used in the experiment are also difficult for the L1 Malay and Malaysian English speakers  and the target words , the mean SNR of the maximum volume level (100) was -8.7 dB. In the first trial, the volume of the multi-talker babble was set to 0 and the amount of change (step size) was set initially at 64. For every reversal, the step size was divided by 2 so that the amount of change in volume decreased across trials. A reversal was defined as a correct response followed by an incorrect response or vice versa. If the response in a trial was correct, the volume was increased by 1 x the step size, if it was incorrect, it was decreased by 3 x the step size. For instance, the multi-talker babble volume increased from 0 to 64 (SNR = -4.8 dB) after the first correct trial. In the second trial, the volume was increased by another 64 but capped at 100 (SNR = -8.7 dB) when the response was correct again. However, if the response was incorrect in the second trial, the volume decreased by 96 . Thus, the volume was reduced from 64 to 0 because the minimum volume level was 0. The minimum step size was 1. The staircase procedure stopped after 12 reversals. At the end of the staircase procedure the final level of background multi-talker babble (threshold level) was set at the average of the volumes from the last 4 reversals. The adaptive staircase procedure used stimuli from the same speaker that was used in the following training session. Importantly, no feedback was provided in the adaptive staircase procedure.As in Experiment 1, participants performed the Same-Different discrimination task with feedback during the training. The only difference with Experiment 1 was that the background multi-talker babble was set at the level determined by the adaptive threshold procedure before the training session.F = 51.42, p < .001.Similar to Experiment 1, pre-test performance differed widely between participants (accuracy range from 53% to 94%). The adverse levels used during training (SNR) were calculated across participants for each speaker and a one-way repeated measure ANOVA was conducted to compare the SNR levels between speakers. Results showed that the male Southern Irish English speaker was least intelligible  compared to the other male Irish English speaker  and the female American English speaker , The mean accuracies of the pre- and post-tests are presented in The participants' identification accuracy before and after training was examined in a 3 x 2 x 2 mixed ANOVA with time of test (pre- vs. post-test) and phonemic contrast  as the within-subject factors and L1  as the between-subject factor.F = 42.50, p < .001, η2p = 0.54, 95% CI, η2G = 0.10. A main effect of L1 was found, F = 4.42, p < .05, η2p = 0.20, 95% CI, η2G = 0.12. Tukey’s HSD post-hoc tests showed a significant lower identification accuracy for the Mandarin group  relative to the Malaysian English group , p < .05. Furthermore, a marginally significant interaction was observed between time of test and L1, F = 3.12, p = .06, η2p = 0.15, 95% CI, η2G = 0.08, indicating that improvements observed potentially differed between the language groups. Post-hoc paired-samples t-tests with Bonferroni correction revealed that Mandarin, t(13) = 4.56, p = .001, gav = 1.03 and Malay, t(13) = 4.53, p < .001, gav = 0.82 groups improved significantly in post-test, whereas Malaysian English group did not. Identification accuracy for stimuli of the /ε/-/æ/ and the /t/-/d/ final contrasts improved similarly because no interaction was found between phonemic contrast and the time of test. Furthermore, there was no three-way interaction between time of test, L1 and phonemic contrast and no other significant effect were found.Overall perceptual performance improved significantly after training , Similar to Experiment 1, training generalization from trained to untrained /ε/-/æ/ stimuli was examined in a 3 x 2 x 2 mixed ANOVA  = 46.20, p < .01, η2p = 0.56, 95% CI, η2G = 0.11. Importantly, no interaction was found between stimuli set and time of test, indicating that perceptual improvements were the same for trained and untrained stimuli. Again, a main effect of L1 was found, F = 3.65, p < .05, η2p = 0.17, η2G = 0.11. Tukey’s HSD post-hoc tests revealed that overall identification accuracy of the Mandarin group  was lower than the Malay group , p < .05, but only marginally different from the Malaysian English group , p = .10. No interaction was found between L1 and time of test, although numerically there were differences between the groups in term of their improvements from pre- to post-test .The analysis revealed again a higher identification accuracy in post-test  compared to pre-test , The HVPT-AAC improved the participants’ perceptual performance by 7% after a total of just one hour of training. The improvements were modulated by English proficiency because larger improvements were found for L1 Mandarin (9.5%) and L1 Malay speakers (8.1%) relative to L1 Malaysian English speakers (3.1%) whose English proficiency was higher than the other two groups. Importantly, the training improvement across groups generalized from trained to untrained /ε/-/æ/ stimuli and to stimuli of the untrained /t/-/d/ final contrast.To compare the effectiveness of HVPT in fixed adverse conditions (Experiment 1) with HVPT-AAC (Experiment 2), the identification accuracy of the L1 Mandarin speakers from Experiment 1 and 2 were analyzed together. Data from the groups with low and high levels of background multi-talker babble of Experiment 1 were collapsed because there was no significant difference in training between these two conditions. To ensure that training effects of both experiments were not affected by differences between the participant groups, the linguistic background and pre-test performance of the participants were compared between Experiment 1 and 2. Between-subject t-tests confirmed that participants of both experiments did not differ in terms of their self-rated English proficiency, self-rated Mandarin proficiency, IELTS standard scores, AOA, and most importantly, their pre-test identification accuracy.F = 41.07, p < .01, η2p = 0.51, 95% CI, η2G = 0.001. Importantly, an interaction was found between time of test and type of adverse conditions, F = 11.11, p < .01, η2p = 0.22, η2G = 0.02 (see t(13) 4.56, p = .001, gav = 1.03 and the fixed level of adverse condition, t(27) = 3.28, p < .01, gav = 0.28. An additional between-subjects t-test was conducted to further examine the interaction. The t-test comparing the improvement size (difference in identification accuracy between pre- and post-test) between the two training conditions showed greater training benefits for HVPT-AAC (10%) compared to HVPT with a fixed level of adverse conditions (3%), t(18.17) = 2.86, p < .01, gs = 0.92. Thus, the adaptive nature of the adverse conditions during training enhanced the effectiveness of HVPT. An important question is whether the training results were long-lasting. This was investigated in the Experiment 3.The mean percentage of correct identification between Experiment 1 and 2 was examined in a 2 x 2 x 2 mixed ANOVA with type of adverse conditions (fixed vs. adaptive) as the between-subject factor, and time of test (pre-test vs. post-test) and phonemic contrast  as the within-subject factors. The analysis revealed improved perceptual performance in post-test  compared to pre-test , 0.02 see . Post-hoNumerous training studies have shown that perceptual training results in long-term benefits ,15,17,19All fourteen L1 Mandarin speaking participants from Experiment 1  and thirteen  out of the twenty-eight L1 Mandarin speaking participants from Experiment 2  returned to complete a second post-test 6 months after the last testing session.t(25) = 2.17, p < .05, gs = 0.8. This AoA difference was unexpected and likely due to a difference in instruction between the experiments and the subsequent interpretation of the term in the questionnaire by the participants. Participants in Experiment 1 interpreted the term as the age first exposed to the language through listening, whereas participants in Experiment 2 interpreted the term as the age first starting to learn speaking English.The linguistic background of these two groups of participants see  was compThe stimuli, design and procedure were identical to the pre- and post-tests used in Experiments 1 and 2.F = 29.30, p < .01, η2p = 0.54, 95% CI, η2G = 0.08. Furthermore, the interaction between time of test and type of adverse conditions revealed again that training in adaptive adverse conditions was more effective than training in a fixed level of adverse conditions, F = 5.31, p < .05, η2p = 0.18, η2G = 0.02  compared HVPT with a fixed level of adverse conditions , t(20.82) = 2.35, p < .05, gs = 0.86.The mean identification accuracies of the delayed post-test after 6 months are presented in 0.02 see . Post-hoF = 3.80, p = .06, η2p = 0.13, 95% CI, η2G = 0.04. Thus, overall identification performance did not significantly reduce 6 months after the immediate post-test.To examine retention of the training improvements after 6 months, a 2 x 2 x 2 mixed ANOVA with time of test (post-test vs. 6-months delayed post-test) and phonemic contrast  as the within-subject factors, and level of adverse conditions (adaptive vs. fixed) as the between-subject factor. This analysis revealed no significant effects or interactions, except for the marginal effect of phonemic contrast, F = 11.30, p < .01, η2p = 0.31, 95% CI, η2G = 0.05. There was a trend towards an interaction between time of test and phonemic contrast, F = 3.25, p = .08, η2p = 0.11, η2G = 0.01, which could be driven by the difference in improvements achieved between the /ε/-/æ/ and /t/-/d/ final phonemic contrasts (see t(26) = 4.76, p < .01, gav = 0.38. However, no significant difference in identification accuracy for untrained /t/-/d/ stimuli was found between pre-test  and at the 6-months delayed post-test .Next, the identification accuracy in the pre-test and the 6-months delayed post-test were examined. This analysis revealed again that perceptual performance in the 6-months delayed post-test  was higher than in the pre-test, asts see . Post-hoF = 24.04, p < .001, η2p = 0.49, 95% CI, η2G = 0.09. There was a trend towards an interaction between the time of test and the type of adverse conditions, F = 3.37, p = .08, η2p = 0.12, η2G = 0.01. Additional between-subjects t-test revealed that after 6 months the perceptual improvements with training in adaptive adverse conditions  was only marginally larger than training in a fixed level of adverse conditions , t(25) = 1.84, p = .08, gs = 0.68. No other effects or interactions were found, which suggests that after six months the training benefits still generalized from trained to untrained /ε/-/æ/ stimuli.The generalization of the training effects from trained to untrained /ε/-/æ/ stimuli were examined using the data of the pre-test and the 6-months delayed post-test. A mixed ANOVA see  revealedThe delayed post-test revealed that perceptual improvements were retained 6 months after training as participants performed similarly in the delayed post-test as in the immediate post-test. The analysis that compared the data of the pre-test with the delayed post-test after 6 months still showed significant perceptual improvements, which was mainly driven by the /ε/-/æ/ stimuli. Further analyses revealed that relative to the pre-test, the delayed post-test after 6 months again showed evidence of generalization from trained to untrained /ε/-/æ/ stimuli. Importantly, there was some evidence that the retained training effect on /ε/-/æ/ stimuli was larger for training in adaptive adverse conditions than training in a fixed level of adverse conditions.The present experiments revealed that after a total of just one hour of high variable phonetic training with English phonemic contrasts in adverse listening conditions, the perceptual performance of Malaysian multilinguals significantly improved. Moreover, the perceptual improvements were successfully retained six months after training, demonstrating sustainability of this rapid and effective training paradigm. Consistent with previous findings, the training benefits generalized to novel stimuli of the trained English contrast, /ε/-/æ/ and to novel speakers , 6, 44. Before discussing the implications of the present findings, it is important to note that the moderate to high English proficiency of the current participants presents actually a challenge for the evaluation of HVPT-AAC because the performance of some participants was already close to the ceiling level in the pre-test. Thus, any training improvements observed with HVPT-AAC are therefore likely to be an underestimation of the impact that the training might have with less proficient participants.rock-lock and road-load continuums. Therefore, phonetic learning might have been more straightforward than in other training studies that involved high variability training stimuli. Nevertheless, we do not rule out the possibility that the perceptual improvement might have induced some learning through repetitive exposure to the stimuli and/or background babble.Similar to previous training studies that compared different training paradigms or training effects between different populations , 44, 56,Importantly, we found greater benefits of HVPT-AAC (Experiment 2) when the adverse conditions level was adapted for each participant than of HVPT with fixed adverse conditions (Experiment 1). The benefits of HVPT-AAC can be attributed to its adaptive ability to individualize the training level to participants with varied pre-test performance, as well as to the training stimuli used in each training session/day that were produced by different speakers. As reported in Experiment 2, we found that the intelligibility of the three speakers used during training differed. This is consistent with the findings by Peng and Wang  who showThe variability of perceptual training studies in the literature in terms of training methodologies  makes comparisons across studies difficult. Some studies reported very large perceptual improvements after training but they usually involved a substantial amount of training time. For example, Richie and Kewley-Port  reportedTo be able to compare the perceptual improvements in our study to studies in the literature involving only non-native speakers and natural English speech stimuli, we calculated for the current and previous training studies the normalized percentage of improvements by dividing the percentage of perceptual improvement (largest percentage reported in each paper) by the number of training hours. The normalized percentages are presented in As can be seen in The high variability training in HVPT-AAC has likely contributed to its effectiveness see , 13–15),–15,13–15Another key factor of HVPT-AAC that has likely contributed to the effectiveness is the use of adverse conditions during training. The adverse conditions make it more difficult to identify the spoken words and stressed the speaker's auditory system so that more attention had to be used to detect relevant acoustic cues. Our results indicate that merely increasing the level of adverse conditions did not impact the perceptual improvements. Critically, it was the adaptive nature of the adverse conditions in HVPT-AAC, which were determined for each participant before each training session, that lead to the highest identification improvements. Unlike HVPT-AAC that involved multi-talker babble to degrade target speech, Iverson, Hazan and Bannister  altered As predicted by Best’s Perceptual Assimilation Model  and KuhlA successful training paradigm should train participants to perceive in a more native-like manner by directing them to the critical acoustical cues the native speakers attend to. When trained participants learnt how to acquire sufficient acoustical information to cue accurate judgments, the reliance and interference of L1 could be reduced. Participants in the present experiments could have learnt to acquire acoustical information from the stimuli of different English varieties  and successfully transferred the learning to identify phonemic pairs of another English variety (British English). This type of learning transfer could indicate the effectiveness of HVPT-AAC by re-directing participants’ attention in perceptual tasks rather than by developing highly detailed representations of the trained speech materials.In today’s society, English serves as a global lingua franca for communication in business and academic settings between speakers with different L1 backgrounds , 60. In S1 FileEnglish minimal pairs used in the experiments.(DOCX)Click here for additional data file.S2 FileParticipants' percentag of correct identifications for the conditions in each experiment.(XLSX)Click here for additional data file.


Training resulted in an activity decrease in regions known to be involved in verbal WM , indicating that the brain became potentially more efficient after the training. These activation decreases were associated with a significant performance improvement in the n-back task inside the scanner reflecting considerable practice effects. In addition, there were training-associated direct effects in the additional, external verbal WM task , and indicating that the training generally improved performance in this cognitive domain. These results led us to conclude that even at advanced age cognitive training can improve WM capacity and increase neural efficiency in specific regions or networks.Neural correlates of working memory (WM) training remain a matter of debate, especially in older adults. We used functional magnetic resonance imaging (fMRI) together with an n-back task to measure brain plasticity in healthy middle-aged adults following an 8-week adaptive online verbal WM training. Participants performed 32 sessions of this training on their personal computers. In addition, we assessed direct effects of the training by applying a verbal WM task before and after the training. Participants (mean age 55.85 ± 4.24 years) were pseudo-randomly assigned to the experimental group ( Working memory (WM) is a capacity-limited cognitive system which is responsible for not only temporally storing information but also manipulating it . ResearcThere have been studies since 2002 aiming at investigating the effects of WM training, showing that WM can be improved when adequate training procedures are used . MoreoveWe investigated a group of healthy middle-aged volunteers within a limited age range . The inclusion of this age group should minimize the influence of relevant age-related changes, such as atrophy or amyloid plaques, while maximizing the usefulness of the training with regard to training gains. We also avoided the inclusion of subjects with cognitive impairment and cognitive complaints, which are preclinical cognitive declines associated with dementia . The selThe aim of this study was to investigate the behavioral and neural changes following an adaptive online verbal WM training in healthy middle-aged participants between 50 and 65 years old. We expected to provide evidence for neural plasticity and/or improvement in behavioral performance in healthy adults within this specific age range.Sixty-three subjects participated in the study. Six participants had to be excluded due to different reasons: one subject dropped out after the first session, two participants had clinically relevant alterations in brain structure, one volunteer moved more than 3 mm during the task-fMRI, one subject’s scanning data was not completely saved, and one participant was a training outlier. Therefore, the final sample contained fifty-seven healthy right-handed volunteers  ranging between 50 and 65 years . Subjects were recruited via advertisements in the internet or newspaper. First, a telephone interview was conducted to assess the basic inclusion criteria: right handed, no mental disorder and presence of metal in the body. Afterward, the following diagnostic checklists were performed: the short form of the geriatric depression scale (GDS) , the minp = 0.91, p = 0.89, p = 0.31, respectively). The study was approved by the Ethical Committee of the Klinikum Rechts der Isar and the Federal Office for Radiation Protection.Written informed consent was provided by each subject before the first session. Study participation was remunerated. Assignment of participants to one of the two groups  occurred pseudo-randomly taking into account gender, age and years of education (YOE). The experimental group included 30 participants , the control group consisted of 27 participants . There were no significant differences between both groups regarding age, sex or YOE . Both groups performed two different WM training modalities: verbal and visual n-back task. Given that the regions involved in verbal and visual WM processes are known to differ and considering that the visual n-back training differed significantly from the verbal training  results of the visual training are reported elsewhere.We used the n-back task as WM training paradigm, in which letters are presented sequentially and the subject is asked to press a key whenever the current letter is identical to the one that appeared n-back positions earlier in the sequence. The active control group performed a low-level vWM training . The vWM training of the experimental group was based on an adaptive online n-back paradigm comprising 9 blocks per session adapted from In the scanner, subjects likewise performed a visual and a verbal n-back task. As already mentioned, visual WM results will be reported elsewhere. The WM paradigm was explained to the subjects before entering the scanner. In addition, subjects were asked to perform a short training version of the task to familiarize themselves with the stimulus presentation. Participants were allowed to repeat the practice task until they reported that they fully understood the task. The vWM task comprised the presentation of 26 capital white letters from the alphabet on a black background in the form of a block design. The whole task consisted of seven blocks of control condition  and seven blocks of active task condition  presented in random order. Each condition lasted 45 s and consisted of 5 s of an instruction display indicating the following condition in German (3-back or X-back/0-back), 5 s of a fixation cross presentation, and 35 s of presentation of the letters see . Each blIn order to investigate potential direct effects of the vWM training we asked participants to perform the HAWIE-R digit span sub-test (forward and backward version)  before a1 and IBM SPSS Statistics software  to analyze the fMRI behavioral data and the HAWIE-R test data. Two different statistical programs were employed to double-check the correctness of our results. Python version 3 was used to analyze the training data and scipy. stats was the package used for the statistical analyses. For the fMRI behavioral data we conducted two repeated-measures analyses of variance (ANOVAs) with Group  as between-subjects factor, Session (S1 vs. S2) as within-subject factor, and mean reaction time or d’ values during each condition (3-back or X-back) as dependent variable. We selected d’ instead of accuracy values [hits – false alarms (FA)] because this parameter takes the range for both components into account by calculating the relative proportion of hits minus FA  to test whether there were any baseline differences between the groups. For the HAWIE-R subtest we likewise conducted repeated-measures ANOVAs with Group  as between-subjects factor and Session (S1 vs. S2) as within-subject factor.We used JASPminus FA . Higher d’ values. As data from the last three sessions of one subject in the experimental group were lost, we interpolated the missing data with her own previous training data with a forward linear method. T-tests comparing the first four and the last four sessions were performed to investigate whether there was a significant improvement in training performance in both groups.For the training data, we analyzed the mean n-back level achieved in each session as well as the ® software 2. The participants were able to see the task through a mirror fixed to the head coil which reflected the MRI-compatible screen. Participants were positioned supinely in the scanner. Their responses were collected via fORP 932 subject response package (Cambridge Research Systems). Participants held the button-box in their right hand and the emergency button in their left hand.There were two scanner sessions: one immediately  before the 8 weeks online training (S1) and another one immediately  after the training (S2). The WM paradigm was presented using Presentation∗-weighted EPI sequence with the following parameters: 237 slices; TR = 2700 ms; TE = 30 ms; flip angle = 90°; voxel size = 3.0 × 3.0 × 3.0; slice thickness = 3 mm; 0.6 mm gap; FOV = 192 mm; interleaved acquisition. The same sequences were used in S1 and S2.Images were acquired on a 3 T Biograph MR-PET Siemens scanner , equipped with a 16-channel head coil at the Klinikum rechts der Isar, Munich, Germany. Specific cushions were used to prevent head movement. The imaging protocol included the following sequences: T1 MPRAGE, T2, FLAIR, DTI, echo-planar imaging (EPI) resting state, task-fMRI, and FDG-PET. Scan sessions lasted approximately 1 h. Results of the other sequences  will be reported elsewhere. A high-resolution MPRAGE anatomical sequence was acquired with the following parameters: 160 slices; TR = 2300 ms; TE = 2.98 ms; flip angle = 9°; voxel size = 1.0 × 1.0 × 1.0; slice thickness = 1 mm; no gap; FOV = 256 mm; interleaved acquisition. Functional data were obtained using a gradient-echo T23 in MATLAB v2018b. First, we performed head motion correction. Here the functional images were realigned and resliced to fit the mean functional image and then co-registered to the MPRAGE image using normalized mutual information. Movement was visually checked for each participant and participants moving more than 3 mm maximum displacement were not included in the final dataset. For the final dataset (n = 57) we calculated the root mean squared head position change (RMS movement) and converted the rotation parameters from degree to mm by calculating displacement on the surface of radius 50 mm to get the frame wise displacement (FD), as reported by Preprocessing as well as statistical analysis of fMRI data were conducted with SPM12 A general linear model at the single subject level was conducted to obtain the task activation contrasts of interest. The task design function was convolved with a canonical haemodynamic response function (HRF) and its time derivative, allowing for a slight temporal shift. Six motion realignment parameters and motion censor regressor  were included as covariates of no interest. We used a high-pass filter of 220 s to the functional data to eliminate low-frequency components because the default filter (128 s) was not adequate for our design .t-test to obtain areas activated during the n-back task (3-back > X-back level) in general. We also performed a two-sample t-test to examine whether there were differences at S1 between the experimental and the active control group. The longitudinal analyses were performed by assessing the interaction effects between Session (S1 vs. S2) and Group  using the factorial design in SPM. The statistical criterion was set at p < 0.05 false-discovery rate (FDR) corrected. In addition, the number of expected voxels per cluster was used an as an extent threshold.For the second level analysis we conducted a one-sample d’ values (both p < 0.001) when comparing performance between the first and the last four training sessions. In the control group, only d’ values were analyzed, since the n-back level  stayed the same during all training sessions. Expectedly, d’ values of the control group did not significantly differ between the first and last four training sessions (p = 0.184).As is illustrated in SD = 0.41) at S1 and 6.89 (SD = 0.33) at S2. Those for the experimental group were 7.77 (SD = 0.39) at S1 and 8.83 (SD = 0.32) at S2. The repeated measures ANOVA on the HAWIE-R forward subtest showed a non-significant effect of Session  but a significant main effect for Group . The interaction between Session and Group was significant  and no effect of Group . The interaction between Group and Session yielded no significant results .The average HAWIE-R backward subtest values for the control group were 6.85 . The average 3-back d’ values for the control group were 2.73 (SD = 0.53) at S1 and 2.96 (SD = 0.61) at S2. Those for the experimental group were 2.74 (SD = 0.51) at S1 and 3.69 (SD = 0.78) at S2  = 47.03, p < 0.001] and for Group  and, accordingly, the interaction between Session and Group was significant . Post hoc analyses revealed no significant improvement in the control group (p = 0.06), but a highly significant improvement in the experimental group (p < 0.001).The comparison between experimental and active control group yielded no significant differences at baseline (S1) in any condition for t S2 see . The repd’ values of 4.18 (SD = 0.13) and 4.08 (SD = 0.19) at S1 and S2, respectively, whereas the experimental group had a mean of 4.10 (SD = 0.29) and 4.13 (SD = 0.21) at S1 and S2, respectively  = 0.93, p = 0.34] or Group . The interaction was also not significant  indicating no performance improvement for the X-back condition in any group after the training.For the X-back condition the control group had mean vely see . The repp = 0.646 and X-back: p = 0.531). Mean reaction time (RT) 3-back for the control group was 782.7 ms (SD = 183.75) at S1 and 713.04 ms (SD = 172.31) at S2, whereas the experimental group had a mean RT of 805.71 ms (SD = 191.67) at S1 and 567.35 ms (SD = 155.75) at S2  = 42.1, p < 0.001], no effect of Group , and a significant interaction between both factors . Post hoc analyses revealed a significant improvement from S1 to S2 in the control group (p = 0.0017) as well as in the experimental group  in any condition for mean reaction time  at S1 and a mean RT of 403.21 ms (SD = 72.32) at S2, whereas mean RT in the experimental group was 458.62 ms (SD = 66.3) at S1 and 428.06 (SD = 60.53) at S2  = 22.51, p < 0.001] but no significant effect for Group . There was also no significant Session by Group interaction . This means that both groups improved after the second session. Post hoc analyses revealed that both the control group (p = 0.002) as well as the experimental group (p = 0.002) improved from S1 to S2.In the X-back condition, the control group had a mean RT of 446.93 ms (t S2 see . The rept test to investigate the brain regions activated in the n-back task (3-back > X-back) independent from training revealed wide-spread cortical as well as subcortical activity . The analysis yielded no significant differences. This means that we can interpret the differences between the groups at S2 as differences arising from the training. All results were p < 0.05 FDR corrected.We also performed a two-sample Experimental Group (S1 > S2) > Control Group (S1 > S2) in mainly superior frontal and parietal regions   as between-subjects factor and Session (S1 vs. S2) as within-subject factor investigating the effects of the cognitive training in both groups for 3-back vs. X-back showed significant results for the interaction ions see . The revrus) see . The revIn the present study, we applied task-fMRI to investigate neural and behavioral effects of an 8-week adaptive online vWM training in middle-aged healthy subjects. We found no differences in brain activity during the n-back task between the experimental and active control group at baseline. Comparing both time points the results showed no activation differences in the control group, but a significantly decreased activation in vWM characteristic regions in the experimental group after the training. These activation decreases, most probably reflecting training-associated gains in cerebral efficiency, were accompanied by significant vWM performance improvements in the experimental group.The general  activation in a predominantly fronto-parieto-cerebellar network that we found by analyzing activation of the whole group at the first timepoint is largely in line with previous studies investigating vWM . HoweverAdaptive online vWM training resulted in reduced brain activity in several parietal areas, first and foremost in the left supramarginal gyrus (SMG), which has been found to be important for the phonological store component, although the exact neural basis of this WM component is still under debate . We alsoThese results are consistent with previous neuroimaging studies that show decreased activation in regions involved in WM processing following cognitive training . Criticad’ values for the vWM condition  whereas there was no such improvement in the low-level X-back condition demanding merely attentional processes. Considering that the training was an adaptive WM training this result is according to expectation. Interestingly, mean reaction times in the 3-back condition decreased in both groups, with the experimental group, however, improving to a considerably larger extent. Taking into account that motor response was practiced in both trainings, this result is likewise in line with our expectations. The performance improvement in the vWM condition from the fMRI task  in the experimental group was backed up by a significant training performance improvement of this group. This means that the improvement manifested itself both in the n-back task performed on the home-computer as well as in a different environment  with a stable n-back level - a clear indication of practice effects. Moreover, the experimental group improved their HAWIE-R digit span forward  performance compared to the control group thus demonstrating direct effects on a similar vWM task. Hence, the training had the expected effects on vWM performance. These results imply that the training was an effective and adequate method to improve WM-relevant processes . The finding that there were no significant improvements in the digit span backward test could be due to the fact that this subtest is significantly more complex than the forward version. Considering that the vWM training did not possess this level of complexity the lacking significance in the backward version is in line with recent results suggesting that the effects of WM training tend to be restricted to the cognitive demands provided by the training  led to significant activation decreases in WM-relevant regions and considerable improvements in vWM performance. In correspondence with the concept of “lifelong learning” present results clearly indicate that neural plasticity and behavioral improvement following vWM training is possible not only at younger age, but also in middle-aged adults.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the Ethical Committee of the Klinikum Rechts der Isar. The patients/participants provided their written informed consent to participate in this study.ME, IY, and KK contributed to the conception and design of the study. ME designed the stimuli and online training, programed the tasks, analyzed the neuroimaging data and training data, and wrote the first draft of the manuscript. ME and IR recruited, scanned, and tested the participants. IR analyzed part of the task-fMRI behavioral data and revisited all different versions of the manuscript. QW analyzed the cognitive tests. KK wrote sections of the manuscript. All authors contributed to the manuscript revision and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Increasing evidence demonstrate that cadmium (Cd) has adverse effects on the mammalian reproductive system. However, the mechanisms underlying the effects of Cd on ovarian function and the strategies to reverse these effects have not been fully elucidated.P value < 0.05 indicated statistical significance.In this study, 60 CD-1 mice were divided into four groups . During the treatment for 14 days, body weight was measured every 2 days. After the treatment, ovaries were isolated and weighted to observe the morphological and biological characteristics. Statistical analyses were performed using one-way ANOVA followed by Fisher’s-multiple range test or chi-squared test, A We observed that Cd exposure induced ovulatory dysfunction, demonstrated by the reduced number of ovulated oocytes numbers in the Cd group. However, this endoplasmic reticulum (ER) pathway was activated in the Cd-exposed ovaries and the expression of GRP78, ATF4, CHOP, and p-JNK was upregulated, which was reversed by treatment with melatonin. Furthermore, we found that melatonin inhibited Cd-induced activation of cleaved caspase-3, restored the ratio of Bax/Bcl-2, and ultimately decreased the apoptosis of granular cells as detected by TUNEL staining.Collectively, our findings reveal that melatonin attenuated Cd-induced ovulation dysfunction and cell apoptosis by inhibiting the activation of the ER pathway. Thus, melatonin can be a potential agent to protect mammalian ovaries against Cd toxicity. Cadmium (Cd) is a ubiquitous environmental contaminant, which cannot be degraded . ExposurMammalian ovaries contain oocytes at different stages of development, and somatic cells of various types. Ovulation is a periodic event, the number of ovulated oocytes strongly associated with female fertility . IncreasMelatonin (N-acetyl-5-methoxytryptamine), a hormone secreted mainly by vertebrate pineal gland. Peripheral tissues such as retina, gut, ovary, and placenta can also produce this indoleamine hormone –19. MelaThe aim of this study was to investigate the detail mechanism of Cd-induced ovary dysfunction, and the possible protective effect of melatonin on the ovary. We found that Cd activated the ER pathway, which ultimately resulted in cell apoptosis in the ovary. These conditions were partly alleviated by melatonin, which suggests the potential clinical application of melatonin in the protection of ovarian function.Female CD-1 mice (5-week-old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . All the animals were kept under a temperature- and humidity-controlled condition on a 12- h light/dark cycle. They were allowed to acclimatize for a week before treatment and had ad libitum access to food and water. All the experiments have received the approval of institutional review board and the First Affiliated Hospital of Zhengzhou University.2, 5 mg/kg; Sigma, St. Louis, Mo, USA) with or without melatonin [2 was dissolved in saline (5 mg/kg).Sixty CD-1 female mice were randomly assigned to four groups. The mice were administered intraperitoneal injections of the vehicle or, melatonin , cadmium chloride   and stained with hematoxylin and eosin. Images were captured with the Nikon Ni-E microscope. To assess the ovulation function, pregnant mare serum gonadotropin  was intraperitoneally injected to stimulate the ovaries of the mice, followed by injection of 7.5 I.U. human chorionic gonadotropin (hCG) after 48 h to induce super-ovulation. After 12–14 h, the mice were sacrificed and the oviductal ampullae were broken to obtain the cumulus-oocyte complexes (COCs). Subsequently, COCs were pipetted in the in-vitro fertilization (IVF) medium (Vitrolife Sweden AB) containing 5% human serum albumin (Irvine Scientific) and 0.1% hyaluronidase (Sigma) to remove the cumulus cells. The number of ovulated oocytes was counted under a stereoscopic microscope .At least three ovaries were homogenized in the lysis buffer from the protein extraction kit . A protein quantitation kit (Bio-Rad) was used to measure the concentrations of the protein solutions. Briefly, the protein samples were separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane. The membranes were blocked with 5% defatted milk in Tris-buffered saline containing Tween20 (TBST) for 1 h at room temperature. Next the proteins were incubated with primary antibodies- GRP78 , ATF4 , CHOP , phosphor-JNK , Bax , Bcl-2 , and cleaved caspase 3 , overnight at 4 °C respectively. Anti-GAPDH monoclonal antibody  was used as a loading control. After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, the proteins were detected by enhanced chemiluminescence detection system (Bio-Rad)., Germany), and the synthesis of cDNA was performed with a reverse transcription kit  according to the manufacturer’s protocol. Real-time PCR was performed using the SYBR Green PCR kit  and Quantstudio 12 K Flex (Applied Biosystems). The primers sequences of GRP78, ATF4, s-XBP1, CHOP, GAPDH are shown in Table -ΔΔCT method. Each sample was measured in triplicate in each experiment.Total RNA was extracted by using the micropurification kit  was purchased to carry out TUNEL analysis based on the manufacturer’s instructions. After incubation in TUNEL reaction mixture for 1 h at 37 °C, the sections were washed twice and counterstained with Hoechst 33342 (10 μg/mL) for 10 min to stain the nuclei. Finally, the sections were mounted on slides and the images were captured under a fluorescence microscope .p value < 0.05 indicated statistical significance.Data are presented as mean ± SD. All the data were analyzed by one-way ANOVA followed by Fisher’s-multiple range test or chi-squared test, using the SPSS 17.0 Package . A The body weights of the mice were recorded every other day. After treatment for 14 days, the ovaries of each group were harvested, and weighed with an electronic weighing balance. Relative ovary weight was calculated as a proportion of the final body weight before euthanasia, expressed as mg/g body weight. The results showed that the Cd-treated group had significantly less body weight than the control group from the 4th day of the treatment. Melatonin ameliorated the effect of Cd on body weights  in the ovaries of different groups (Fig. GRP78, ATF4, CHOP comparing to the control ovaries, and tended to have higher expression of sXBP-1. Notably, administration of melatonin was able to reduce the ER stress in Cd-treated mice comparable to the controls (Fig. ER stress has been demonstrated to be a key mechanism underlying Cd-induced cell death. To elucidate the molecular mechanisms underlying the alleviation of ovulation disorders in mice by melatonin, we carried out the western blot assay, and the results showed that the expression of the proteins— GRP78 and ATF4 was significantly upregulated in ovaries of Cd-exposed mice. However, melatonin notably attenuated the Cd-evoked upregulation of GRP78 and ATF4 Fig. a and b. ups Fig. c. The reols Fig. a. These Increased ER stress induced cell apoptosis by activating the caspase pathway; hence, we examined the protein expression of cleaved caspase-3, Bax and Bcl-2 in the ovary by western blotting. Results showed that the protein expression of cleaved caspase-3 indeed substantially increased in Cd-exposed ovaries. Meanwhile, administration of melatonin attenuated the cleaved caspase-3 level indistinguishable from the controls. The Cd group showed a higher level of Bax and a lower level of Bcl-2 protein than the control. Melatonin reversed the negative effects of Cd treatment on the expression of Bax and Bcl-2 proteins. We assessed the Bax/Bcl-2 ratio; results showed that the ratio of Bax/Bcl-2 was considerably higher in the Cd-exposed ovaries than in the ovaried of the mice in the control group. The elevated ratio was reversed by melatonin (Fig. Cd exposure is associated with female infertility and in vitro fertilization outcomes in humans , 36. In XBP-1—an important downstream molecule of the IRE1 pathway, was also increased in the ovaries of Cd-exposed mice. The protein and the mRNA levels of GRP78, ATF4 and CHOP were significantly upregulated, indicating the activation of the PERK pathway. These results were consistent with previous findings that Cd affects the reproductive system by elevating the level of ER stress in chicken ovary [ER is sensitive to the disruption of cellular homeostasis. Perturbed ER function, known as “ER stress”, is able to trigger the UPR to restore cellular homeostasis , 13. Earen ovary . Anotheren ovary . A recenen ovary . All theExcessive ER stress is related to cell apoptosis. CHOP and p-JNK are proapoptotic proteins that could promote the expression of apoptosis-related proteins , 15. TheTaken together, our study demonstrated that melatonin can ameliorate Cd-induced ovulatory dysfunction and ovarian injury by suppressing ER stress, suggesting that administration of melatonin has a protective effect against ovary damage caused by Cd exposure. However, additional studies are still needed to investigate the modulatory pathway.


The developed films were characterized by scanning electron microscope and evaluated for thickness, weight variation, and elasticity. The developed films showing optimal mechanical properties were selected to evaluate radial swelling properties, in vitro release of metronidazole and the antimicrobial activity against Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans by the disc diffusion method. The results demonstrated that LM-MZ and HM-G-MZ films were colorless and yellowish color, respectively, with the film thickness around 0.36–0.38 mm. Furthermore, both films exhibited good elasticity with low puncture strength  and also showed slight increase in radial swelling, so that they could be easily inserted and fitted into the periodontal pocket during a clinical use. However, HM-G-MZ showed a decrease in radial swelling after 1 h due to the film erosion. The in vitro release study of LM-G-MZ showed a burst release that was initially followed by a slow release rate profile, capable to maintain the therapeutic level in periodontal pocket for seven days, whereas HM-G-MZ showed an immediate release profile. The cumulative percentage of metronidazole release from HM-G-MZ was less than LM-G-MZ during the first 5 min as metronidazole was in a crystalline form inside HM-G-MZ film. For antimicrobial activity test, both films showed the inhibitory effect against P. gingivalis and A. actinomycetemcomitans, and there was no difference in the inhibition zone between LM-G-MZ and HM-G-MZ. The present study showed, for the first time, that low methoxyl pectin film containing glycerin and metronidazole could be potentially considered as a promising clinical tool for the drug delivery via intra-periodontal pocket to target an oral disease that is associated with polymicrobial infection.The objective of this study was to develop the metronidazole loaded high and low methoxyl pectin films (HM-G-MZ and LM-G-MZ) for the treatment of periodontal disease. The films were prepared by pectin 3%  Periodontal disease is one of the most prevalent oral diseases affecting more than 30% of Thai community . It is aGardnerella vaginalis [Since periodontal disease is associated with polymicrobial infection, antibacterial treatment such as metronidazole might be useful for improving the condition of the inflamed tissues. Metronidazole is a partially hydrophilic drug, which effectively inhibits an infection of anaerobic microorganisms and protozoa ,6. It isaginalis ,8,9.dl-lactic acid) (PDLLA) and poly(dl-lactide-co-glycolide) (PDLGA) was developed. The films demonstrated the controlled-release profile of metronidazole lasting longer than one-month for treating periodontal pocket infections [However, there are still limited approaches used for preparing the formulations of sustained/controlled-release metronidazole films aided in an intra periodontal pocket application. In 2009, metronidazole-loaded bioabsorbable films consisting of different ratios of poly was purchased from Du Pont®, Wilmington, DE, USA. Metronidazole was purchased from Alfa Aesar, Tewksbury, MA, USA. Calcium chloride (CaCl2) and disodium hydrogen phosphate were purchased from Merck . Distilled water served as the solvent for preparing film solutions. All of the reagents were analytical grade.Non-amidated low methoxy pectin  was purchased from CargillPorphyromonas gingivalis strain ATCC 33277 and Aggregatibacter actinomycetemcomitans strain ATCC 43718 were obtained from the stock collection of the Oral Biology Research Division, Khon Kaen University. Both bacteria were grown in Wilkins-Chalgren Anaerobe broth  consisting of tryptone (10 g/L), yeast extract (5 g/L), menadione (0.5 mg/L), and hemin (5 mg/L). A culture of P. gingivalis was incubated in a 3 mL broth overnight in a 37 °C anaerobic condition that was achieved by using AnaeroPack®-Anaero  in combination with a leak-proof jar, otherwise maintained on blood agar plates. A. actinomycetemcomitans was grown in a similar broth but under a CO2 condition.Two different species of periodontal pathogen were utilized for testing the ability of metronidazole-containing films to statically inhibit bacterial growth, as well as to kill periodontal microbes. w/v of HM in distilled water at 60 °C for 1 h and allowed for complete hydration and swelling overnight, after which 5% metronidazole together with or without glycerin 40% w/v of polymer was added to HM solution, gently stirred using a glass rod, and left until all of the air bubbles disappeared and casted in a horizontally leveled plastic plate. For LM, film formulations comprising of 3% w/v LM with or without glycerin 40% w/v and metronidazole 5% w/v were prepared and casted on a 6 cm × 6 cm dialysis membrane . Then, the casted-film forming solution on the membranes were placed on crosslinking agent (3% w/v CaCl2) supported by plastic box for 2 h [HM or LM pectin films containing metronidazole were prepared while using conventional casting or modified ionotropic gelation techniques, respectively. Sample code and film compositions were shown in The morphology was examined while using scanning electron microscopy (SEM) with a JEOL scanning electron microscope  at 10 kV under low vacuum mode. The film characterizations were performed without any coating solution at magnifications of ×150. The thickness and surface of films were evaluated.The thickness of each 3 cm × 3 cm sized film was measured at 10 points while using a thickness gauge . Ten films in circle shape with 4 mm diameter were individually weighed by analytical balance . The mean film thickness (in mm) and weight (in mg) with the standard deviation were calculated. 2). Needle stainless probe (2 mm) was used (probe contact area = 3.14 mm2). The texture analyzer was adjusted for the probe’s forward movement at a velocity of 1.0 mm/s. Measurement started when the probe had contacted the sample surface (triggering force). The probe moved on at constant speed until the film was torn. The applied force and displacement (penetration depth) were recorded. All of the experiments were conducted at a room condition . The mechanical strength of the film was characterized by maximum applied force (Max. force), displacement, area under the force-displacement curve (AUG), and puncture strength [Mechanical strength of the films was tested using a texture analyzer TX.TA plus . An individual sample holder has been constructed to facilitate measurements of a 3 cm × 3 cm sized film samples. Film was fixed by screws between two plates with a cylindrical hole of 9.0 mm diameter /D0. Where %Sd is the percentage swelling that is obtained by the diameter method, Dt is the diameter of the swollen disc after time t, and D0 is the original dry disc diameter.Swelling behavior of metronidazole-loaded pectin films was evaluated by the diameter method . Six fil2) of metronidazole into 2 mL of phosphate buffer, (pH 6.6) under a constant stirring at 400 rpm for 4 h until complete disintegration. The dispersion was filtered and the absorbance measured at 318.5 nm by UV spectroscopy . Metronidazole content was determined from the standard curve of metronidazole in buffer, which was linear with a high correlation coefficient (r2 = 0.9994). The following regression equation was obtained: y = 0.0541x + 0.0138, where y is the absorbance and x is the concentration of metronidazole (µg/L). The experiment was done in triplicate. The percentages of drug content were calculated.The amount of metronidazole loaded into the selected film (LM-G-MZ and HM-G-MZ) was determined by adding a 4 mm diameter film containing 146.7 µg (11.66 µg/mmw/v w/v) was added to prevent contamination by various microorganisms. At the different time intervals, 0.5 mL samples were collected at 5, 10, 15, 30, and 60 min for HM-G-MZ and until seven days for LM-G-MZ. The samples were diluted 20 times before measured at 318.5 nm by UV spectroscopy. All of the dissolution runs were performed in triplicate.Metronidazole-loaded pectin films with a diameter of 4 mm were immersed in 2 mL Tris buffer gingival fluid pH 6.6 at 37 °C up to seven days in semi static conditions to determine the kinetics of metronidazole release from the pectin films. Sodium azide  at 600 nm wavelength and further diluting the cultures to obtain the OD of 0.1 by using sterile TYKH broth. A 100 µL prepared bacterial culture was spread on top of the blood agar plate prior to placing pectin films containing metronidazole. To ensure the killing ability of metronidazole against two tested periodontal pathogens, we also performed the experiment to determine the minimum inhibitory concentration (MIC) of metronidazole and incorporated on the blood agar plates. Briefly, two-fold dilution of metronidazole was made using ultrapure water to achieve the dose range of 0.16 to 5 mg/mL. A 10 µL metronidazole at each concentration was dropped on the sterile 6 mm disc separately. In addition, we used the disc containing 10 µL ultrapure water as a negative control. Metronidazole-loaded pectin films were subsequently placed on the blood agar inoculated with each bacterial strain. Our test samples were incubated under the optimal conditions for bacterial growth for 48 h until a lawn of bacterial colonies was clearly seen. The diameters of an inhibition zone in which the width of the film was included were measured while using Mitutoyo® Digimatic caliper . Three independent experiments were performed in triplicate.p-value < 0.05, unless otherwise stated. Statistical analysis was performed while using SPSS software version 16.0 All data are presented as mean ± SD. One-way ANOVA was used to evaluate the significance of differences at All of the developed films were transparent and odorless. All types of LM films were slightly yellowish to colorless, while HM films provided yellowish to light brown color . Generaln = 10 per group) demonstrated a slight difference of thickness and weight (Determination of film thickness and weight variation was performed to ensure the consistency of film preparation as well as the amount of the drug in the film. The results showed that the comparison between different type of custom-made films (d weight . Notablyp < 0.05). The effect of glycerin on the properties of pectin film was similar to another study demonstrating that, even though the addition of glycerin leads to the reduction of a static modulus, dynamic modulus, and tensile strength, the elongation of the films was found to be increased [2 but not significantly increased in HM-MZ. While, adding glycerin in metronidazole-loaded film showed a significant decrease in the mechanical properties (p < 0.05). For example, the puncture strengths of LM-G and LM-G-MZ were 2.05 and 1.63 N/mm2, whereas puncture strengths were 1.37 and 0.84 N/mm2 for HM-G and HM-G-MZ, respectively. Interestingly, the reduction of mechanical strengths in high methoxyl pectin containing metronidazole films (HM-MZ and HM-G-MZ) was higher than those that were comprised of low methoxyl pectin (LM-MZ and LM-G-MZ). This discrepancy might be contributed by the film network and the difference of crystal state of metronidazole in LM and HM. This result is consistent with a previous study of Preis et al. reporting that amorphous state provided higher mechanical strength. Due to the strong interaction of molecules, rigidity and large size of metronidazole, crystal state provided steric effect as a barrier between the interaction of polymer chain. While in amorphous state, the interaction between drug molecules was lower than crystalline form, known to be less affected by the interaction between polymer chain [The result of mechanical strength of metronidazole pectin films was shown in ncreased . Adding er chain .One crucial factor of film formulations is the mechanical strength of the films during the development and production process. In this study, metronidazole films without glycerin (LM-MZ and HM-MZ) were very brittle and easily broken when cut into small circular shape prior to use in further experiments. Therefore, HM-G-MZ and LM-G-MZ films were chosen, since they provided superior mechanical properties. LM-G-MZ film slightly swelled 0.8% within 60 min a, maintaLM-G-MZ film showed a fast metronidazole release at the initial stage, up to 60.26% within 5 min followed by a slow release rate from 60% to 80% in 60 min a. In addP. gingivalis and A. actinomycetemcomitans and found that the growth of P. gingivalis and A. actinomycetemcomitans was inhibited at the concentration of 5 mg/mL and 2.5 mg/mL, respectively. This suggests the killing selectivity of metronidazole on different bacterial species. Next, we investigated the efficacy of our LM and HM films in killing two tested bacterial strains. Test films were sterilized by ethylene oxide prior to testing the anti-bacterial activity. The results of the antimicrobial activity of the films against the growth of P. gingivalis and A. actinomycetemcomitans for 48 h using agar disk diffusion method are shown in P. gingivalis; 28.09 ± 1.88 and 31.28 ± 2.05 mm for A. actinomycetemcomitans, respectively. The rapid HM-G-MZ film dissolution might be involved in the slightly greater clear zone of HM-G-MZ than LM-G-MZ film, as shown in grey bars of P. gingivalis and A. actinomycetemcomitans have been considered to be major periodontal pathogens that are associated with severe and chronic periodontitis [P. gingivalis and A. actinomycetemcomitans.We first determined the MIC of metronidazole in killing dontitis ,27,28. IP. gingivalis and A. actinomycetemcomitans, the HM-G-MZ and LM-G-MZ films showed similar inhibitory effect. This study indicated that LM pectin film prepared by ionotropic gelation technique in which intramolecular cross-links occurred between negatively charged carboxyl groups of LM and the positively charged of cross-linked solution was suitable for sustained-release drug dosage forms. Whereas, HM pectin film using the solvent casting method might be more appropriate for an immediate-release product. These results could be useful for further study to investigate the mucoadhesive properties, as well as human skin irritation, so as to contribute to further development of these thin films containing antimicrobial agent for eliminating a microbial infection in the periodontal pockets.In this study, pectin films that were loaded with metronidazole were prepared and evaluated for their morphology, mechanical properties, loading content, metronidazole release and anti-bacterial activity. Addition of glycerin in pectin film formulations decreased the maximum applied force and increased the displacement, which appeared to improve the film flexibility when compared to the films designed without glycerin. All of the pectin film formulations exhibited fairly uniformed drug content with 100% metronidazole. Rapid dissolution was observed in HM-G-MZ film. However, LM-G-MZ film showed a fast release rate at the initial stage, followed by a slow release rate that is capable of maintaining approximately 90% release up to seven days. According to the test of antimicrobial activity against


The results show that all metal decorations can improve the adsorption strength of phosphorene to nitrogen-based gases molecules except for Tl decoration. Especially, the adsorption energy of NH3 molecule on Ni decorated phosphorene is 1.305 eV, and the adsorption energies of NO and NO2 on La decorated phosphorene can be up to 2.475 and 3.734 eV, respectively. In addition, after NO and NO2 adsorptions, the electronic and magnetic properties of some metal decorated phosphorenes change, indicating that the metal decorated phosphorenes have great potential in NO and NO2 detection.Using the first-principles calculation based on density functional theory (DFT), the adsorption properties of nitrogen-based gases molecules (NH Thus, the removal and detection of nitrogen-based exhaust gases from environment is essential. Traditionally, metal oxide semiconductors have been widely used to detect of nitrogen-based gases due to low cost, low power consumption and simplicity in measurements9. However, their long recovery period and low specificity limit the application in high measurement accuracy gas detection10.Nowadays, many human activities, such as burning, motor vehicle exhaust, and chemical production processes, produce polluting gases. Among many atmospheric pollutants, nitrogen-based gases  decorated phosphorene were studied systematically. It is found that all metal decorations can improve the adsorption strength of nitrogen-based gas molecules on phosphorene, except for NO molecule on Tl decorated phosphorene, and thus metal decorated phosphorenes have great potential for application in nitrogen-based gas removal. Furthermore, the electrical and magnetic properties of some metal decorated phosphorene have interesting change after NO and NO37. The generalized gradient approximation (GGA) with the parametrization of Perdew-Burke-Ernzerhof (PBE) was adopted38. The empirical correction scheme of Grimme (DFT + D2) was included to correct the effective van der Waals (vdW) interaction39. The kinetic energy cutoff was set to be 500 eV for all calculations. The 3 × 3 × 1 mesh grid was sampled in 1st Brillouin zone by using Monkhorst-Pack scheme. To avoid the interaction between periodic adsorbates, the 3 × 4 supercells were employed. A vacuum of 15 Å was used perpendicular to the phosphorene layer to ensure that the adjacent phosphorene sheets do not interact with each other. For structural optimization, the force convergence criterion on each atom in the structure is less than 0.01 eV/Å. The adsorption energies (Eads) of nitrogen-based gas molecules on pristine phosphorene (bP) and metal decorated phosphorene (bP-M) are calculated byEtotal is the total energy of the adsorption system. EbP−M and Eadsorbate are the energies of bP/bP-M and nitrogen-based gas molecule, respectively.All the density functional theory (DFT) calculation have been implemented by using the Vienna ab initio simulation package (VASP)40. For the nitrogen-based gas molecules  on pristine phosphorene, we consider three highly symmetric adsorption sites  of the adsorption systems were calculated as shown in Fig. 2 molecules and phosphorenes are larger as compared with NH3 adsorbed case, implying the larger interaction between the NO/NO2 molecules and phosphorenes and thus larger adsorption energies. The charge transfer quantity between the NH3 molecule and phosphorene is very small, indicating that the interaction between NH3 and phosphorene is weak, which is consistent with the minimum adsorption energy. In addition, the Bader charge analysis indicates that the electrons transfer from phosphorene to NO and NO2 molecules are 0.242 and 0.300 e, respectively, while the electrons transfer from NH3 molecule to the phosphorene is 0.030 e. Thus, the NO and NO2 molecules act as acceptors, whereas the NH3 molecule acts as a donor, which is consistent with ref.22.The optimized lattice constants along the armchair and the zigzag direction are 13.722 and 13.225 Å, respectively, and the bandgap is 0.88 eV for 3 × 4 monolayer phosphorene supercell with 48 phosphorus atoms, which are consistent with the ref.2 and NH3 molecules on the electronic properties of phosphorene, the band structures and local density of states (LDOSs) of the adsorption systems were calculated, as shown in Fig. 2 molecules introduce spin polarization in phosphorene, while the NH3 adsorption system is of zero magnetic moment , alkaline earth metal , transition metal  and post transition metal (PTM = Tl)3 molecules adsorption on bP-M systems, the various initial adsorption configurations are considered. The most stable adsorption configurations are obtained by comparing the adsorption energies after full geometry optimization. The most stable adsorption configurations are illustrated in Fig. Eads) are listed in Table 3 molecules are stably adsorbed on bP-M systems with N atoms pointing toward the metal. The adsorption energies range from 0.238 to 1.305 eV for all the stable adsorbed cases, which are larger than that of 0.218 eV for adsorption on pristine phosphorene, indicating that the decoration of metal can improve the adsorption energy of phosphorene to NH3 molecule. In addition, the charge transfer amounts (∆Q) and adsorption distances (DP-Gas/M-Gas) between NH3 and metal decorated phosphorene are also summarized in Table 3.For the NH3 molecules adsorption on bP-M systems. The adsorptions of NH3 molecules have little effect on the electronic structures of bP-Ms due to that the states of N and H atoms are far below the Fermi level. As shown in Fig. 3 adsorption are consistent with those without NH3 adsorption, which are also corroborated by the band structures in Fig. 3 adsorption doesn’t change magnetic property of bP-M adsorption systems.Figure 3 molecules. Except for bP-Sr, the conductive properties of the other bP-M system remain unchanged after NH3 adsorptions. For example, the bP-AM , bP-Ba and bP-Tl adsorption systems still remain metal properties, while the bP-Ca, bP-Ni and bP-La adsorption systems still remain semiconductor properties with the corresponding bandgaps of 0.045, 0.788, and 0.188 eV, respectively. For bP-Sr adsorption system, as shown in Figs 3 adsorption. In addition, the overlap between the coupling peaks of N and H and the metal indicates strong hybridized interaction between them, which may be the cause that the metal decoration can improve the adsorption strength of phosphorene to NH3 molecules. However, for the NH3 molecule adsorption on the bP-Tl system, except for the N and H coupling peaks near −4 eV in Fig. 3 molecule and T1 atom.The Fermi levels of the decorated substrates shift slightly due to the certain amount of electron transfer between the decorated substrates and NHThree different adsorption configurations were modeled for NO adsorption on bP-M: the N-O bond is parallel to phosphorene plane and N-O bond is normal to phosphorus plane with N or O pointing to metal atom. The results show that the NO molecule prefers the N atom to be close to the metal atom due to the larger adsorption energy, as shown in Fig. Figure 43.From Fig. As mentioned above, except for Tl decoration case, all the metal decoration can increase the adsorption energies of NO adsorption on phosphorene, and thus improve the adsorption of phosphorene to NO molecules. For the bP-AM adsorption system, the states of AM atoms distribute mainly in conduction bands as mentioned before, and thus the peaks, near VBM and in the conduction bands, from N and O atomic states hybridized with those of AM atoms. For the bP-AEM adsorption system, the spin-up states in bandgap from NO molecules overlap significantly with those from AEM atoms, which account for the larger adsorption energies of NO adsorbed bP-AEM systems. For the LDOSs of NO adsorpted bP-Ni as depicted in Fig. 2 molecule is adsorbed on the bP-Ni system with the bond angle away from Ni atom, while the remaining NO2 molecules are stably adsorbed on the metal decorated substrates with the bond angles toward metal atoms. Similar to NO molecules, the adsorption energies of NO2 molecules on bP-AM and bP-AEM systems decrease with increasing the atomic number of decoration atoms, and the adsorption energies of NO2 on bP-AEM are larger than those on bP-AM  adsorption on metal  decorated phosphorene has been studied by using first-principles calculation based on DFT. Except for NO adsorption on bP-Tl, all metal decorations can improve the adsorption strength of phosphorene to nitrogen-based gas molecules. For NH3 and NO, the adsorption energies follow an energy order of Eads (bP-TM) > Eads (bP-AEM) > Eads (bP-AM) > Eads (bP-Tl), while the NO2 follow an energy order of Eads (bP-La) > Eads (bP-AEM) > Eads (bP-AM). The largest energies of NH3, NO and NO2 adsorbed bP-M are 1.305, 2.475 and 3.734 eV, respectively. In addition, the band structures and LDOSs show that NH3 has little effect on electronic and magnetic properties of bP-M substrates, while the adsorptions of NO and NO2 make an interesting change in electronic and magnetic properties of bP-M. For example, except for bP-La, the metal decorated substrates exhibit nonmagnetism-to-magnetism after NO adsorption, and the bP-Ca, bP-Sr and bP-La undergo semiconductor-to-metal transition. Similarly, the magnetic states of some decorated substrates  change after NO2 adsorption, and especially the spin polarization for bP-La systems disappear after NO2 adsorption. Furthermore, the bP-Li, bP-Na and bP-Tl experience metal-to-semiconductor transition, while bP-Ca and bP-Sr undergo semiconductor-to-metal transition due to large electron transfer amounts between NO2 and decorated substrates. These results indicate that the metal decoration can be used for the removal and detection of nitrogen-based gas.In conclusion, the nitrogen-based gas molecules (NHSupporting Information